In the time since our last blog post we have tried several attempts to perform a successful conjugation with multiple strains of Pseudoalteromonas and Planococcus associated with the accessory nidamental gland of the squid. We follow the conjugation protocol for the conjugation. Conjugation is how bacteria exchange genetic information. In this case our E. coli conjugates contain a transposon, which basically randomly inserts into DNA and we are hoping for this transposon to insert in the codon that codes for biofilms for our bacterial samples. Here are the basic steps from this protocol: first overnight cultures of or samples are grown. The next day we combine our intended recipient bacteria with the E. coli conjugant that has the transposon and pellet them using a centrifuge. We then resuspend the pellet and then spot the mixture of the two on a plate. We then let the bacteria grow over night at room temperature where the two should conjugate. The next day we scrape the spot off the plate, resuspend in Luria broth and pipette 10-50 microliters onto fresh plates and spread it evenly using sterile glass beads. These plates are then grown at room temperature overnight.
There is a selective antibiotic marker for erythromycin that is combine into the recipient bacterium during the transposition. This selective agent allows for us to grow up our transformed bacterial colonies. What we are hoping to get for results include interruption of the genes for biofilm and pigment formation. We will be screening for colonies with no biofilms and impaired pigment formation. However we have had difficulties with the bacterial conjugation and we have not had high numbers of bacterial transformants. Which means for some reason our bacteria isn’t conjugating with E. coli and we are trouble shooting this process.
We have recently ordered fresh antibiotic to add to the plates which should limit the growth of the donor E. coli but allow for our transformed bacteria to grow. One other factor that can be optimized to increase transformation is increasing the moisture content of the media. If this next round is not successful we may need to reorder our conjugate strains.
Over this semester we have made a lot of progress on our research. We started by finalizing the literature review and methodology portions of our paper and right now we are at the stage of gathering all the data. Right after getting the research approved by IRB two weeks ago I started interviewing students on the Pleasantville campus. It has been a great experience full of learning. Since this is the first time I conduct interviews at the beginning knowing how to remain neutral throughout the interview was a big challenge. However, by sticking to the questions that we have drafted and some practice, I have been able to learn some strategies to make sure the question I am asking are as neutral as possible.
Over the last few weeks, I have learned more than I thought. A very valuable lesson has been understanding how to get people to discuss topics that are usually avoided in social settings. Since our research is focused on identifying behaviors and perceptions towards community service and civic engagement, the role of politics and government plays is significant and usually avoided by people. For my surprise, all the students that I have interviewed have reacted in a very positive way and have been willing to share all their opinions very openly despite how strong they are.
It is now a critical time in my research process. After working on developing a thorough literature review and seeing what similar research has shown, it is time for me to analyze the results from my survey. I have never done research quite as extensive as this so I am both excited to see the results and what they will dictate, as well as nervous. I know I have full support from my advisor but it is still an intimidating concept to analyze research like this when you have never been in this sort of situation. I am ready to dive in though, and I know that this will be a learning and growing experience for me.
As the deadlines for my research are growing closer, I know the critical nature of this time in my research process is so important. This is the time where I will truly discover if all of the time I have spent wondering what my results will yield will finally come to light. I downloaded the results from my survey today and will begin analyzing them over the course of the next few weeks. I look forward to this extensive process and cannot wait to put my final results into my report. I am ready to share with my community what I have discovered.
Since my last blog post, I have made promising progress with my research project. My main goal of this semester was to focus on microscopy. Now that I have established that my gene of interest, F10C2.4, clearly shows some kind of reproduction problem when not present using RNA interference, I wanted to utilize the unique feature of my model organism, Caenorhabditis elegans, to determine what is going wrong in the organisms without this gene. One of the drawbacks from using technology such as a microscope hooked up to a computer is that sometimes things can go wrong; however, I feel that I have learned so much from the mistakes, which has helped me get a better understanding of the research I do.
Caenorhabditis elegans have many benefits for genetic manipulation and research. One of the most beneficial features is that it is transparent. This is great for microscopy because it makes it easier for us to see what is different with the worm’s reproductive system when comparing it to the normal, not treated worm. For the experiments I perform for the microscopy element, we repeat the RNAi interference experiments with strains with fluorescent markers. GFPs are green fluorescent proteins that can stain a particular part of a cell; like a cell wall and RFP are red fluorescent protein can stain the chromosomes within the nucleus of the cell. With the strain I am working with, AJ740, I can utilize the GFP and RFP to see what is happening to the shape and overall placement of the eggs within the affect mother worm treated through RNA interference along with what is going on with the chromosomes. I have several questions. General questions like: are the eggs going to the right place and are there too many or too little eggs in the mom’s body? In regards to the chromosomes, are they separating at the right time? Is there an issue that is causing the incorrect amount of chromosomes in the cell which is not allowing them to hatch once they are laid? Does the egg go through the correct amount of cell divisions before being laid? All these questions can be answered using microscopy thanks to the organism, C. elegans.
A challenge that I have been facing, along with other members in my research group, is in regards to getting the microscopy aspect to work. This has opened my eyes to the many components involved in a task like this where things can go all good for one week but the next week, multiple things can go wrong. That being said, it is important to troubleshoot what went wrong and come up with ways to fix the problem at hand. An example of this is when the GFP and RFP do not show up bright enough to identify anything going on in the C. elegans. This could be due to the organisms not growing properly due to a lack of food or temperature sensitivity. Whichever the case, it was important for us to work together as a team to come up with a way to avoid this issue for the next week. For the future, I hope I can take more images from microscopy to look further into what is going on internally to determine what the function is of my gene of interest, F10C2.4.
For my research on gentrification, I have begun interviews with people impacted by the recent changes in Brooklyn. The goal is to bring a qualitative element to the project to show how real people are dealing with displacement and the changing demographics. We will ask if they know anyone has been displaced or if has impacted them in any way, what subtle changes they notice around them, and how they feel about the overall trend. How, if at all, is Brooklyn different now than it was a decade or two ago? We have an understanding of the policies and events that brought New York from a financial crisis to the economic powerhouse it is now, but what does it look like on the ground for the people who witnessed the transformation? A difficulty we are having is coordinating schedules and finding room in people’s schedules.
For Voces, we are collecting more essays from Latinxs in the New York area. We want to showcase the broad spectrum of experience and show there is not one singular Latinx narrative in the United States. I am writing a piece for it and a difficulty I am having is trying to properly word my experiences in a way that is meaningful for the reader. Some of the new authors are having similar difficulties finding ways to identify and write about issues that are personal to them.
My research on the Density Functional Theory and Raman spectroscopy of ten different sulfonamide antibiotic derivatives has really been coming along. I have now completed the assigning of the different vibrational modes for all of the bonds in all ten of the antibiotics. This part of the experiment has taken me the longest to complete. As I was creating the graphs of the Raman spectra data, I noticed that my signal to noise ratio was a little low. This means that the peaks weren’t intense enough compared to the background interferences. To improve the signal to noise ratio, I ran my samples three more times each under the same conditions. Then I used to average of all three runs to plot new graphs with more intense data.
Being a part of this project has taught me a lot so far. Not only have I become quite comfortable using the portable Raman spectrometer that we have here at Pace and using the computer program Gaussian to analyze the theoretical data but I have learned that running an experiment requires a lot of patience. A lot of my time is spent waiting for the samples to finish a run and then I am able to analyze it. It also requires a lot of thinking on your feet because not everything goes as planned and you have to be ready for that.
I have finally been able to create a poster to show the results that I have obtained so far. The next step is to assign the functional groups in charge for the molecular vibrations at the particular peaks.
I believe as time has passed, I’ve made some good progress, especially since my last blog post. I used to struggle the most with injecting my fish embryos with morpholino oglionucleotide, as well as using the confocal microscope. However, now I find both techniques easy and rather fun. This was a big challenge I overcame and now feel confident doing. By being able to do both these crucial steps in my study, I’ve started to collect some very interesting data. I’m seeing that at some specific concentration of morpholino, under the confocal microscope there seems to be an increase in the amount of hair cells within each neuroblast. This was very exciting for both my mentor and I.
Finding the concentration of the morpholino to inject was rather hard. Often times my injections were killing the embryos because the concentrations were too high. Many trials needed to be done to find the right amount to inject. One of the biggest things I’ve learned through this experiment was to be patient. I never expected it to take so much time. However, it was my patience and practice that finally allowed me to get good and promising results. My next step in my study is to kill off the hair cells in my fish by use of copper treatment in order to measure the rate of growth and cell number of the hair cells.
Since the last update, I have finished doing the wet lab portion of my experiments. My mentor has taken my samples and gotten the CD spectra for my samples. The next step for me is to analyze the results given from the results. I will be doing that by creating graphs in IGOR and analyzing the CD using an online program provided by my mentor. I was focusing on looking at the percent difference in the alpha helices and beta pleated sheets before and after the binding.
Over the break I have made most of the paper that I was planning to do. I was able to read many literature on my topic. I was able to do most of the thesis, the only things left to put are the final analyzed data and the discussion of the results and future works. I have made my poster and put it out for printing and am preparing to present it in two weeks for the ACS conference before I present it at the UGRI research day in May.
For the rest of this project my goals are to finish analyzing the results using the online program and computer program stated above, finish the paper, and to practice speaking about my project so that I will be prepared for the poster sessions coming up.
The Study right now is moving along slowly but surely. We have continued our work with coding our current transcriptions. while reviewing our current codes we have decided to clean up our data and consolidate it further. We continue to conduct new interviews and finish transcribing until we reach our number of saturation. Our current number of participants is about 31.
As we continue our work with this qualitative research I recognize how patient one must be with the work. I have learned a lot in the world of coding and in the world of research. Most recently my realization of how much time studies actually take and how patient you must be with yourself and your work is something I am learning.
In the previous blog post, I walked you through the manner in which I would set up my next experiment. In that experiment, I exposed five N2 L4 hermaphrodites to three treatments; L440 (negative control), npp- 19 (positive control), and M05D6.2 (gene of interest), using RNAi. From that experiment, what I was able to conclude was that the M05D6.2 treatment did not have a significant impact on N2 hermaphrodites in regards to fertility. This finding helps support my claim that the M05D6.2 gene does not impact hermaphrodite fertility but does impact male fertility. The progeny counts for the M05D6.2 and L440 treatments were very similar. To show the significance of this data I will use the program R to perform statistical analysis on the collected data.
Along with collecting this data, I performed microscopy on male C. elegans, using the strain AJ740. Prior to viewing the worms, they were exposed to an RNAi treatment using the gene M05D6.2. This strain was used due to the fact that they have a GFP tag on their tubulin, allowing them to fluoresce when being viewed under a fluorescent microscope. What I was looking for within these males was a decrease in sperm production. From the images that I captured I did see that this strain was able to produce sperm after being exposed to the RNAi treatment, but to properly say if there was a decrease or not in sperm production I will have to repeat this experiment.
In the following weeks, I plan to set up a number of different experiments. One of those experiments is to cross five N2 C. elegans males with spe-8 hermaphrodites. Spe-8 worms cannot properly produce their own sperm, so if a spe-8 animal self-fertilizes it produces a “dumpy” worm as opposed to if it were to mate with a N2 male, producing a normal sized worm. So, if there is a larger count of dumpy worms after the cross then we can say that silencing the M05D6.2 gene causes an impact on male fertility. Because a higher number of dumpy worms means that they hermaphrodite was self-fertilizing as opposed to crossing with the N2 males. Another experiment I want to do is creating another replicate of my initial experiment where I crossed five rrf-3 males (RNAi sensitive males) to a spe-8 hermaphrodite. After setting up these experiments I will pick off the males that were used and DAPI stain them. By DAPI staining the males it will allow them to be viewed under a fluorescent microscope.